PROJECT SUMMARY/ABSTRACT Melanoma, the deadliest form of skin cancer, is characterized by aberrant hyperactivation of the ERK mitogen- activated protein kinase (MAPK) signaling pathway. Inhibitors of BRAF and MEK, both members of this pathway, have shown clinical efficacy and are used in combination to treat BRAF mutant melanoma, the most common melanoma genomic subtype. However, not all BRAF mutant melanomas respond to these inhibitors, and those that do respond eventually acquire resistance. Better understanding the molecular mechanisms underlying the response and resistance to BRAF and MEK inhibitors will bring us closer to developing durable therapeutic strategies. The main goal of this project is to establish a novel regulatory role for PPP6C, the catalytic subunit of protein phosphatase 6 (PPP6C), in the ERK signaling network. This will allow for understanding how observed genetic lesions in PPP6C alter this oncogenic signaling pathway, likely driving malignant transformation as well as drug resistance in melanoma. Our interest in PPP6C is based on 1) preliminary work observing downregulation of PPP6C promotes resistance to MEK inhibition and increases ERK activation and 2) whole exome and targeted sequencing projects identifying PPP6C is mutated in 7-12% of melanomas. This proposal will examine the effects of PPP6C silencing on ERK activation and inhibitor sensitivity across a panel of melanoma patient-derived cells to confirm a role for PPP6C in regulating ERK signaling. The specific signaling mechanisms underlying PPP6C regulation of ERK signaling will be determined by cellular and biochemical approaches investigating how PPP6C directly or indirectly regulates MEK activation. Regulation of ERK signaling and MEK/BRAF inhibitor susceptibility by cancer-associated PPP6C mutations will be examined in cultured cells and in a mouse model of melanoma. To assess the tumorigenic potential of PPP6C loss, PPP6C knockdown melanocytes will be evaluated for growth factor independent proliferation and anchorage independent growth. The findings from these studies will provide insight into how to optimize the use of clinical MEK and BRAF inhibitors in melanoma and identify opportunities to improve the efficacy of targeted therapies.